Genetic linkage has been studied and linkage maps have been developed for a wide variety of species, including plant species. Localization of genes of interest can be accomplished through linkage analysis with mapped markers as described by Patterson, E. B. (1982) "The mapping of genes by the use of chromosomal aberrations and multiple marker stocks", pp. 85-88, In: Maize for Biological Research (W. F. Sheridan, ed.) University Press, University of North Dakota, incorporated herein by reference.
The concept of using markers associated with favorable agronomic traits to track and recover the favorable traits in segregating populations is known to the art, e.g. Atkins et al. (1942), "The isolation of isogenic lines as a means of measuring the effects of awns and other characters in small grains," J. Amer Soc Agron 34:667-668; Everson, et al. (1955), "The genetics of yield differences associated with awn barbing in the barley hybrid (Lion.times.Atlas).times.Atlas," Agron. J. 47:276-280; Carol Rivin et al. (1983) "Evaluation of Genomic Variability at the Nucleic Acid Level," Plant Mol. Biol. Reporter Vol. 1, p. 9; Helentjaris, T. G., PCT Application published Dec. 6, 1984, "Process for genetic mapping and cross-breeding thereon for plants".
Such genetic linkage has been invaluable in the introgression of specific chromosomes or chromosome segments into various genetic backgrounds (Rick, C. M. and Khush, G. S. (1969) "Cytogenic explorations in the maize genome", pp. 45-68, In: Genetics Lectures Vol. I (R. Bogart, ed.), Oregon State University Press, Corvalis; and C. Rhyne (1960) "Linkage studies in Gossypium II altered recombination values in linkage group of allotetraploid G. hirsutum L. as a result of transferred diploid species genes" Genetics 45:673-683). The use of genetic markers speeds the transfer of a specific locus to a desirable genotype. In plant breeding, tissue of young plants can be tested for the presence of marker alleles linked to the desirable trait and only individuals displaying the presence of such marker alleles need be grown to adulthood, transplanted and used to produce progeny, thus eliminating many time-consuming steps required in traditional plant breeding. For example, the tomato nematode resistance gene, mi has been successfully transferred though linkage with an acid phosphatase isozyme marker (Tanksley, S. D. et al. "Use of an Acid Phosphatase Isozyme for Predictive Association with an Agronomic Trait," Plant Mol. Biol. Rep., In press). Such markers are also useful in facilitating the recovery of a desired recurrent parent in a backcrossing program (e.g. S. D. Tanksley, H. Medina-Filho and C. M. Rick (1981) "The effect of isozyme selection on metric characters in an interspecific backcross of tomato-basis of an early screening procedure" Theor. Appl. Genet. 60:291-296).
Molecular markers such as isoenzyme, protein and nucleic acid markers, the variants of which do not often have any noticeable effect on phenotype are preferred over the phenotypic markers used in classical breeding methods. See Newton, K. J. et al. (1980) "Genetic basis of the major malate dehydrogenase isozymes in maize," Genetics 95:424-442; Goodman, M. M. et al. "Maize", Isozymes in Plant Genetics and Breeding, Part B (Tanksley, S. D. et al. eds.) (1983) Elsevier Science Publishers.
Nucleic acid markers provide certain advantages over isozyme and protein markers. With DNA markers, allelic variation is detected by first digesting DNA from the individuals being analyzed with a variety of restriction endonucleases. The resulting fragments are separated by electrophoresis and transferred to solid support matrices. Allelic fragments are then identified by hybridizing the DNA on the supports to cloned, radioactively-labelled, homologous sequences. Genetic variation detected in this manner has often been referred to as restriction fragment length polymorphism (RFLP). The number of RFLP's are virtually unlimited. They are unlikely to have an effect on phenotype, are codominant and are inherited in a predictable fashion.
A theoretical discussion applying known methods of genetic mapping to RFLP's and practical applications thereof is given in Beckmann, J. S. and Soller, M. (1983), "Restriction fragment length polymorphisms in genetic improvement: methodologies, mapping and costs", Theor. and Appl. Genetics 67:35-43; and Soller, M. and Beckmann, J. S. (1983), "Genetic polymorphism in varietal identification and genetic improvement," Theor. and Appl. Genetics 67:25-33, both of which are incorporated herein by reference. See also Burr, B., Evola, S. D., Burr, F. A. and Beckmann, J. S. (1983), "The application of restriction fragment length polymorphisms to plant breeding", Genetic Engineering Principles and Methods, (Setlow and Hollander, eds.) Vol. 5:45-49, also incorporated herein by reference, and Ellis, T. H. N. (1986) "Restriction Fragment Length Polymorphism Markers in Relation to Quantitative Characters", Theor. Appl. Genet. 72:1-2. The usefulness of RFLP mapping for maize also has been discussed by S. V. Evola et al. (1986) "The suitability of restriction fragment length polymorphisms as genetic markers in maize", Theor. Appl. Genet. 71:765-771. No specific map positions for any DNA probes are discussed in any of the above articles.
Map positions for many cloned DNA sequences have been reported in connection with maize (Zea mays) Helentjaris, T. et al. (1986) "Use of monosomics to map cloned DNA fragments in maize", Proc. Natl. Acad. Sci. USA 83:6035-6039. This article reports the identification of 112 loci using RFLP's. The fragments mapped by Helentjaris et al. are defined relative to their relationship to certain previously-mapped markers, and relative to each other. This article is incorporated herein by reference. Other mapping efforts are currently in progress throughout the industry and the maize genome is rapidly becoming saturated with mapped molecular markers which are freely available to the public.
While nucleic acid (RFLP) markers have been used to locate and manipulate traits determined by single genes, they have not been successfully used to locate and manipulate traits determined by more than one gene. Burr, B. and Burr, F. A. (1985), "Toward a Molecular Characterization of Multiple Factor Inheritance," Biotech. in Plant Sci. (Zaitlin, M. et al. eds.) discusses this concept in general with respect to quantitative traits without providing specific enablement. Landry, B. S. and Michelmore, R. W. (1985), "Methods and Applications of Restriction Fragment Length Polymorphism Analysis to Plants," Tailoring Genes for Crop Improvement (Bruening G., et al. eds.) 25-44 is a general review article containing a section discussing the use of molecular markers to track and manipulate quantitative trait loci, but without providing enabling disclosure.
A disadvantage in the use of molecular markers for tracking and breeding traits is the fact that cross-overs occurring in progeny predictably will separate the trait of interest from the linked marker used to track it in a certain percentage of individuals. Nuinhaus, J. et al. (1987), "Restriction Fragment Length Polymorphism Analysis of Loci Associated with Insect Resistance in Tomato," Crop Sci. 27:797-803.
Another disadvantage of prior methods for tracking traits using molecular markers is the fact that a particular linked marker allele may not invariably correlate with the presence of the phenotype being studied. Many phenotypes are developmentally expressed, and unless the populations are scored at multiple times during their life cycles, important associated marker alleles can fail to be identified.
Helentjaris, T. (1987), "A genetic linkage map for maize based on RFLPs," Trends in Genetics 3:217-221 provides a maize linkage map and several loci for plant height determinants with the relative contribution of each loci to the phenotype indicated. No enabling method for determining such loci is provided, however. Edwards, M. D., et al. (1987), "Molecular-Marker-Facilitated Investigations of Quantitative-Trait Loci in Maize. I. Numbers, Genomic Distribution and Types of Gene Action," Genetics 116:113-125, provide a method for locating quantitative trait loci using molecular markers. In this method, single-factor analysis is used to determine loci associated with a number of different traits. This analysis was followed by a multiple regression method to determine the relative contribution of each such locus to the given trait. This method, while identifying loci determining polygenic traits and the relative contribution of each, has the drawback of failing to provide a method for ensuring against loss of the trait being tracked due to cross-over in progeny populations. The method described above also fails to take into account the possibility of developmentally-expressed phenotypes.
Nienhuis, J. et al. (1987), "Restriction Fragment Length Polymorphism Analysis of Loci Associated with Insect Resistance in Tomato," Crop Sci. 27:797-803 discloses the use of RFLP technology to identify quantitative trait loci affecting expression of insect resistance in a wild tomato species. Conventional linkage analysis was used to locate RFLP loci associated with the trait, followed by linear and multiple regression to determine the relative contribution of each locus. Analysis of the residual plots indicated that one or more additional loci with major effects had not been identified. The article suggests the use of flanking markers to localize a target quantitative trait locus, but characterizes this as "problematic."
No previously described method for locating DNA governing polygenic traits has been successfully used to introgress such traits into a second or elite genotype.
The present application provides a method for tracking and manipulating polygenic traits in a breeding program which solves the problem of loss of the trait due to cross-over in the progeny population. This method involves the analysis of molecular marker linkage data for a predetermined polygenic trait by the method of multiple regression by leaps and bounds (Furnival, G. M. and Wilson, Jr., R. W. (1974) "Regression by leaps and bounds," Technometrics 16:499-511). This method was developed to assess the relative contributions of causative factors on effects, (i.e. numerous independent factors on dependent variables), and has not previously been applied to genetic analysis, possibly because of lack of appreciation by those skilled in the art of the possibility of making an analogy between such classical causative factors and marker alleles.
The method of the present application also ensures that marker alleles corresponding to developmentally expressed phenotypes are identified.
The method of the present application is exemplified by the identification of loci determining maize dwarf mosaic virus (MDMV) resistance in maize. Maize dwarf mosaic virus occurs throughout the United States and Europe. Resistant cultivars of dent corn have been developed, but sufficient genetic loci determining such resistance to enable introgression of the trait into a variety lacking such resistance have not been previously identified. In an abstract for a presentation to the 78th Annual Meeting of the American Society of Agronomy at New Orleans, Louisiana Nov. 30 through Dec. 5, 1986, G. E. Scott reports the linkage of MDMV resistance to endosperm color in corn, concluding that one or more genes for resistance must be located on the long arm of chromosome 6. The abstract does not provide an enabling disclosure nor locate the gene or genes with sufficient exactitude to enable their isolation. Resistant cultivars of sweet corn having quality factors acceptable to the industry have not been developed, leading to serious economic losses in the United States due to MDMV. Use of identified loci for MDMV resistance is thus useful for producing inbred cultivars of resistant sweet corn.
Inheritance of resistance to MDMV is not clearly understood. The number of genes which contribute to resistance and the nature of gene action appears to be significantly dependent upon the source of MDMV resistance, the susceptible inbreds, the time of scoring, and the method of inoculum production and application. (Louie, R. (1986), "Effects of genotype and inoculation protocols on resistance evaluation of maize to maize dwarf mosaic virus strains," Phytopathology 76:769-773 .
Roane et al. (1983), "Inheritance of resistance to maize dwarf mosaic virus in maize inbred line Oh7B," Phytopathology 73:845-850, reported that in crosses between the resistant line Oh47b and two susceptible lines, Oh43 and Pa91, the inheritance of resistance was conditioned by one dominant gene. Rosenkranz, E. and Scott, G. E. (1984), "Determination of the number of genes for resistance to maize dwarf mosaic virus strain A in five corn inbred lines," Phytopathology 74:71-76, showed that the inbreds Ga203, Ar254, and Pa405 appear to have three, two and five additive resistance genes respectively. Crosses in which the resistant lines B68 or Pa405 were the donors, and susceptible sweet corns were the recipients revealed three genes, one of which must be present with the other two (Mikel, M. A. et al. (1984), "Genetics of resistance of two dent corn inbreds to maize dwarf mosaic virus and transfer of resistance into sweet corn," Phytopathology 74:467.
The difficulty of assessing genotype from phenotype, and the existence of as many as five significant genes make MDMV resistance an ideal problem for the application of RFLP technology. A further difficulty is provided by the fact that genomic material of resistant MDMV inbred lines tends to move in large segments. This makes it difficult to maximize the presence of genes governing the desired trait from the donor parent while minimizing the presence of surrounding, less desirable DNA. This problem is not specific to MDMV, but is a common problem which is difficult to identify and deal with not only in maize but in the selective breeding of other species as well. The present invention involves the identification of chromosome regions which are associated with MDMV resistance, the prediction of which progeny in an advanced generation will be resistant and which not, and the assessment of recovery of the elite genotype. Rates of convergence upon the desired genotype are significantly increased while risk of losing essential marker loci is substantially reduced.